Biotechnology Explorer

Transcription

1 Biotechnology Explorer Green Fluorescent Protein (GFP) Purification Kit Catalog Number EDU For Technical Service Call Your Local Bio-Rad Office or in the U.S. Call BIORAD ( )

2 A Complete Teaching Guide Developed over five years, Biotechnology Explorer kits and curricula have been written for teachers, by teachers, and have been extensively field-tested in a broad range of classroom settings from high school through the undergraduate level. Easy-to-use Biotechnology Explorer kits are the perfect way to bring the excitement of biotechnology into the classroom. Each kit contains an innovative step by step protocol, which makes them the perfect choice for both experts and beginning teachers. The curriculum contained within the manual for each kit makes our products unique. Each kit contains its own unique curriculum package which is divided into a Teachers Guide and Student Manual. The Teachers Guide is divided into three sections which help to insure that the labs run smoothly. One section contains background information, lecture topics, and suggested references which will enable each teacher, both experienced and newcomers to biotechnology, to prepare and design lectures and lessons which can precede the actual labs. This advance preparation will virtually insure that the labs run smoothly and that the students understand the concepts behind each lab. There is a detailed section on the laboratory set up, complete with simple procedures which contain graphic diagrams detailing the advance preparation for the labs. This makes the set up for each lab virtually foolproof. In addition, this section contains time tables which will help you plan your schedule accordingly. Each lab can be performed in a 50 minute time period, which should fit into most schedules. Finally, we provide a detailed Teachers Answer Guide which contains answers to all of the questions posed in the Student Manual. The teacher can use these answers as a guide when reviewing or grading the questions presented in the Student section of the manual. Each kit is designed to maximize student involvement in both the labs and the thought questions embedded in the manual. Student involvement in this process results in an increased understanding of the scientific process and the value of proceeding into a task in an organized and logical fashion. Students who engage in the science curriculum found in the Bio- Rad explorer kits develop a positive sense of their ability to understand the scientific method. In order for your students to gain the most from this experiment they should know what a gene is and understand the relationship between genes and proteins. For a more detailed discussion of these and other basic molecular biology concepts and terms, refer to the review provided in Appendix B. We strive to continually improve our curriculum and products. Your input is extremely important to us. Incorporation of your ideas, comments, critiques, and suggestions will enable the explorer products to evolve into even better teaching aids. You can find the catalog and curriculum on the internet. Look up our home page at or call us at Ron Mardigian Product Manager, Biotechnology Explorer Program ron_mardigian@bio-rad.com

3 Instructors Guide Table of Contents Page Kit Inventory Check List Kit Components and Required Accessories...2 Suggested Lesson Flow Planning Your Week...4 Workstation Check List Student and Instructor Lab Set-ups...5 Implementation Timeline Advanced Prep and Student Lessons...6 Lesson Highlights Detailed Instructors Guide...9 Quick Guide Graphic Laboratory Protocol...16 Teacher Answer Guide Answers to Student Review Questions...20 Student Manual Green Fluorescent Protein (GFP) Purification...26 Lesson 1 Genetic Transformation Review: Finding the Green Fluorescent Molecule...27 Lesson 2 Inoculation Growing a Cell Culture...28 Lesson 3 Purification Phase 1 Bacterial Concentration and Lysis...32 Lesson 4 Purification Phase 2 Removing Bacterial Debris...36 Lesson 5 Purification Phase 3 Protein Chromatography...39 Appendices Appendix A Glossary of Terms...43 Appendix B Molecular Biology Concepts and Terminology...47 Appendix C Sterile Technique, Working with E. coli and Disposal of Biological Waste...52 Appendix D Gene Regulation and Genetic Selection...53

4 Is Finding a Needle in a Hay Stack Easier When it Glows? This laboratory activity is designed to follow Biotechnology Explorer Kit 1, Bacterial Transformation the pglo System (catalog number EDU). Students begin this activity with the bacteria they genetically transformed using the plasmid, pglo. Transformed bacteria which produce the genetically engineered Green Fluorescent Protein (GFP) are removed from their agar plates and allowed to multiply in liquid nutrient media. The bacterial cells are then broken open (lysed) to release the Green Fluorescent Protein. GFP is subsequently purified from the contaminating bacterial debris using the disposable chromatography columns provided in this kit. The unique fluorescent properties of GFP allow the entire process to be observed using a long wavelength UV lamp (i.e. pocket geology lamp). One of the basic tools of modern biotechnology is DNA splicing, cutting DNA and linking it to other DNA molecules. The basic concept behind DNA splicing is to remove a functional DNA fragment let s say a gene from one organism and combine it with the DNA of another organism in order to make the protein that gene codes for. The desired result of gene splicing is for the recipient organism to carry out the genetic instructions provided by its newly acquired gene. For example, certain plants can be given the genes for resistance to pests or disease, and in a few cases to date, functional genes can be given to people with nonfunctional or mutated genes, such as in a genetic disease like cystic fibrosis. Genes can be cut out of human, animal, or plant DNA and placed inside bacteria. For example, a healthy human gene for the hormone insulin can be put into bacteria. Under the right conditions, these bacteria can make authentic human insulin. When allowed to multiply in gigantic vats (fermenters) these bacteria can be used to mass produce the human insulin protein. This genetically engineered insulin is purified using protein chromatography and used to treat patients with the genetic disease, diabetes, whose insulin genes do not function normally. A common problem in purifying genetically engineered designer proteins from transformed bacteria is contamination by endogenous bacterial proteins. Chromatography is a powerful method used in the biotechnology industry for separating and purifying proteins of interest from bacterial proteins. Proteins purified in this manner can then be used, for example, as medicines to treat human disease, or, for household agents such as natural enzymes to make better laundry detergents. The cloning and expression of the GFP gene (pglo Transformation Kit 1), followed by the purification of its protein in kit 2, is completely analogous to the processes used in the biotechnology industry to produce and purify proteins with commercial value. The real-life source of the Green Fluorescent Protein gene is the bioluminescent jellyfish Aequoria victoria. In this excercise, you may suggest a hypothetical scenario to your students in which GFP has some special commercial value and its gene comes from a different natural source, plant or animal. In either case, the principle is exactly the same, the gene codes for a Green Fluorescent Protein. Bioengineered DNA was, weight for weight, the most valuable material in the world. A single microscopic bacterium, too small to see with the human eye, but containing the gene for a heart attack enzyme, streptokinase, or for ice-minus which prevented frost damage to crops, might be worth 5 billion dollars to the right buyer. - Michael Crichton - Jurassic Park 1

5 Instructors Guide Kit Inventory ( ) Checklist This section lists the components provided in the GFP Purification Kit. It also lists required accessories. Each kit contains sufficient materials to outfit eight student workstations. Use this as a checklist to inventory your supplies before beginning the experiments. Components Provided with the Kit Number/Kit ( ) Ampicillin lyophilized 1 vial Arabinose lyophilized 1 vial LB-broth tablet (to make 50 milliliters) 1 tablet Inoculation loops packs of 10 loops 2 pk Pipettes sterile, individually wrapped 40 Microtubes 2.0 milliliters, clear 30 Culture tubes 15 milliliters, sterile (pack of 25) 1 pk Collection tubes 5 milliliters, polystyrene 25 TE buffer (10 mm Tris, 1 mm EDTA, ph 8.0; sterile) 1 bottle Lysozyme lyophilized 1 vial Binding Buffer (4 M NH 4 SO 4 /TE, ph 8.0) 1 bottle Column Equilibration Buffer (2M NH 4 SO 4 /TE, ph 8.0) 1 bottle Column Wash Buffer (1.3 M NH 4 SO 4 /TE, ph 8.0) 1 bottle HIC chromatography columns 8 Column end caps 1 bag Foam microtube rack 8 2

6 Accessories Not Included in the Kit Number/Class ( ) Transformation plates (Kit 1 LB/amp and LB/amp/ara) 2 UV lamp - Long Wavelength* Bio-Rad catalog number EDU 1 4 Microwave oven 1 1 liter flask milliliter flask milliliter and 250 milliliter graduated cylinder 1 Distilled water (1 gallon container from supermarket) Thermometer 1 Centrifuge 1 Beaker of water for rinsing pipettes 1 Bleach 10 milliliters Magic Markers 1 Refrigerator freezer 1 Optional Shaking/Rocking Platform or Incubator 1 Use of a rocker or shaker will speed bacterial growth in liquid cultures, but is not required. *UV lamp Ultraviolet radiation can cause damage to eyes and skin. Short-wave UV is more damaging than long-wave UV light. The Bio-Rad UV lamp recommended for this module is long-wave. If possible, use UV-rated safety glasses or goggles. 3

7 Suggested Lesson Flow There are five lessons in the GFP Purification Kit curriculum, including four active student laboratory sessions. All lessons are designed to be carried out in consecutive 50 minute periods. For continuity, the five lessons can be conveniently started on a Monday and completed on a Friday. Step by step protocols employed in the student labs are detailed in the Student Manual. Lesson 1 Introduction to Purification. Lecture and discussion. Lesson 2 Picking Colonies and Inoculating Cell Cultures Lesson 3 Purification Phase 1 Bacterial Concentration and Lysis Lesson 4 Purification Phase 2 Removing Bacterial Debris Lesson 5 Purification Phase 3 Protein Chromatography Advance Preparation Instructors Overview This section outlines the recommended schedule for advance preparation on the part of the instructor. A detailed advance preparation guide begins on page 6. Prep Activity When Time required Read manual Immediately 1 hour Prepare Liquid Culture Media Before Lesson 2 15 minutes Set up Workstations Day of each lesson 5 minutes/day 4

8 Workstations Daily Inventory Check ( ) List Student Workstations Materials and supplies that should be present at each student workstation site prior to beginning each lab activity are listed below. The components provided in this kit are sufficient to outfit 8 complete student workstations. Instructors (Common) Workstation Materials, supplies, and equipment that should be present at a common location that can be accessed by all students during each lab activity are also listed below. It is up to the discretion of the teacher as to whether students should access common buffer solutions/equipment, or whether the teacher should aliquot solutions and operate equipment. Lesson 2 Lesson 4 Student workstations Number/team Student workstations Number/team Transformation plates from Microtubes 1 Kit 1 Pipette 1 (LB/amp/ara and LB/amp) 2 Microtube rack 1 Inoculation loops 2 Marker 1 Culture tubes containing Beaker of water for 2 ml growth media 2 rinsing pipettes 1 Marking pen 1 HIC chromatography Test tube holder 1 4 column 1 Column end cap 1 Instructors workstation Shaking incubator or platform 1 Instructors workstation (optional) Binding Buffer 1 vial UV light 1 4 Equilibration Buffer 1 vial Centrifuge 1 UV light 1 4 Lesson 3 Student workstations Lesson 5 Microtubes 1 Student workstations Pipettes 1 Collections tubes 3 Microtube rack 1 Pipette 1 Marker 1 Microtube rack 1 Beaker of water for rinsing Marker 1 pipettes 1 Beaker of water for rinsing pipettes 1 Instructors workstation HIC chromatography TE solution 1 vial column 1 Lysozyme (rehydrated) 1 vial Column end cap 1 Centrifuge 1 UV light 1 4 Instructors workstation Wash Buffer 1 vial Equilibration Buffer 1 vial TE Buffer 1 vial UV light 1 4 5

9 Instructors Advance Preparation for Labs This section describes preparation to be performed in advance by the instructor for the active lab sessions in Lessons 2 through 5. Lesson 2 Inoculation Growing a Cell Culture Lesson 3 Purification Phase 1 Bacterial Concentration and Lysis Lesson 4 Purification Phase 2 Removing Bacterial Debris Lesson 5 Purification Phase 3 Protein Chromatography For students to begin Lesson 2 they will need the two transformation plates (LB/amp/ara and LB/amp) from Kit 1. To avoid contamination, bacterial cells from these plates should be used within 3 days following the transformation activity. At the completion of the transformation activity, store the transformation plates in a refrigerator to keep the cells fresh. Lesson 2 Inoculation Growing a Cell Culture Advance Preparation Objectives Prepare liquid culture media (6 8 below) Set up student and instructors workstations (Page 5) Set up rocking table or shaking incubator or incubator oven Time Required 30 minutes Note: Observe sterile technique while preparing the following materials. See Appendix C. Prepare Ampicillin and Arabinose Solutions Ampicillin and arabinose are shipped dry in small vials. After being rehydrated, both are added to the liquid growth media. Ampicillin is an antibiotic which inhibits growth of bacterial contaminants which may be introduced from the environment. Arabinose is a sugar which induces the overexpression of the Green Fluorescent Protein in cloned cells. Refer to Appendix A and D for more details on the functions of these two components. Using a sterile pipette, add 3 milliliters of TE Solution directly to the vial containing the ampicillin. Using another sterile pipette, add 3 milliliters of TE Solution to rehydrate the arabinose. Mix the vials and gently swirl or vortex to aid in rehydration. 1ml 1 ml TE solution Ampicillin Arabinose Note: Rehydrate ampicillin and arabinose the day you prepare the liquid growth medium. Arabinose requires 10 minutes to dissolve be patient. 6

10 Prepare Liquid Nutrient Media In Lesson 2, each student workstation will require two culture tubes containing 2 milliliters of liquid nutrient media. These will be used to grow bacterial cultures. To prepare the liquid media, add 55 milliliters of distilled water to a 250 Erlenmeyer flask and heat to boiling in a microwave. Then, add the single LB tablet to the flask. Let the tablet soak in the hot water for 20 minutes; this will aid in dissolving. Heat the flask again to boiling in the microwave. Swirl the flask to dissolve the tablet. Repeat heating and swirling several times until the entire tablet is dissolved, but be careful to allow the flask to cool a little before swirling so that the hot medium does not boil over onto your hand. Microwave to boiling Microwave to boiling Swirl Add water Add tablet When the entire tablet is dissolved, allow the LB to cool so that the outside of the flask is comfortable to hold, or below 50 C. While the media is cooling, get the ampicillin and arabinose solutions that were prepared in step 1 above. When the media has cooled, use a new pipette and transfer 0.5 milliliters of arabinose and 0.5 ml of ampicillin into the flask. Swirl the flask to mix the components. You can now discard the residual arabinose and ampicillin solutions. 1ml Add Arabinose and Ampicillin Swirl Aliquot Liquid Culture Media Using a new pipette as above, aliquot 2 milliliters of the liquid media into 16 culture tubes. (This can be accomplished by transferring the media in two 1 milliliter aliquots from a sterile pipette.) Store the culture tubes in a refrigerator until the day of use. Nine extra culture tubes are provided. You may wish to fill these tubes with media and use them for demonstrations or for backup liquid cultures for those student teams that do not have successful growth. For Lesson 2, each student workstation will need two 15 milliliter culture tubes, each containing 2 milliliters of liquid culture media. In this lesson, students inoculate their 2 milliliter cultures with transformed bacteria from Kit 1. 7

11 Note: Vigorous shaking of the liquid cell cultures provides more oxygen to the dividing cells, allowing them to multiply faster. A shaking incubator aids in this process. If such a device is not available, cell cultures may be shaken manually for 30 seconds and incubated at 32 C for 24 hours. Simply have the students shake the capped culture tubes for 30 seconds, as they would a can of spray paint. The more vigorous the manual shaking, the more growth, which results in greater production of GFP. After shaking, lay the tubes on their sides in the incubator oven for 1 2 days. When the tubes are oriented horizontally, more surface area of the culture media is exposed to the air in the tube, allowing more oxygen to diffuse into the cells. Shake periodically during the 2 day incubation. If a rocking table, shaking water bath, or orbital shaker is available, adjust the device to run between 40 to 200 RPM. Again, the more vigorous the shaking the better. Shaking at 32 C will provide sufficient bacterial growth in under 24 hours. Cells will also grow sufficiently at room temperature (with rocking). Stationary cultures are not recommended for room-temperature growth. In either case, following the appropriate incubation period, cell cultures inoculated with white and green transformed colonies should appear bright green under ultraviolet light. Lesson 3 Purification Phase 1 Bacterial Concentration and Lysis Advance Preparation Objective Set up workstations Rehydrate lysozyme Set up centrifuge Time required 10 minutes Rehydrate the vial of lyophilized lysozyme with 1 milliliter of TE solution using a new pipette. Mix gently to aid in the resuspension. Keep the vial of lysozyme on ice or in a refrigerator until use. Lesson 4 Purification Phase 2 Removing Bacterial Debris Objective Time required Set up workstations (the only preparation needed) Set up centrifuge 10 minutes Lesson 5 Purification Phase 3 Protein Chromatography Objective Set up workstations (the only preparation needed) Time required 10 minutes 8

12 Lesson Points to Highlight This section describes steps in the experimental protocols which may be technically challenging or which are extremely important to the overall outcome and understanding of the experiments. Instructors should alert their students attention to these points, and when possible, demonstrate the technique before the students attempt the procedure. The Student Manual contains the detailed descriptions and drawings of all laboratory steps and the techniques employed in Lessons 2 5. Refer to it for questions about the experimental protocols used in lab. Use of the Pipette Before beginning the laboratory sessions, point out the graduations on the pipette to the students. Both the 250 µl and 1 milliliter marks will be used as units of measurement throughout Lessons 2 through 5. 11ml 750 µl 500 µl 250 µl 100 µl Lesson 1 Genetic Transformation Review Growth Media The liquid and solid agar media are referred to as LB (named after Luria-Bertani) broth and are made from an extract of yeast and an enzymatic digest of meat byproducts which provides a mixture of carbohydrates, amino acids, nucleotides, salts, and vitamins, all of which are nutrients for bacterial growth. Agar, which is derived from seaweed, melts when heated and cools to form a solid gel (very analogous to Jell-O), and functions to provide a solid support on which to culture bacteria. 9

13 Antibiotic Selection The pglo plasmid which contains the GFP gene also contains the gene for beta-lactamase, a protein that provides bacteria with resistance to the antibiotic, ampicillin. The betalactamase protein is produced and secreted by bacteria which contain the plasmid. The secreted beta-lactamase inactivates ampicillin, allowing only transformed cells to grow in its presence. Only transformed bacteria which contain the pglo plasmid, and produce beta-lactamase can survive in the presence of ampicillin. (See schematic below.) Bacterial proteins Arabinose promotor protein arac ori bla GFP Bacterial chromosomal DNA Beta-lactamase protein Green fluorescent protein arac ori bla GFP Genetically engineered plasmid used to insert new genes into bacteria. "GFP" Gene which codes for the Green Fluorescent Protein. "bla" Gene which codes for beta-lactamase, a protein which gives bacteria resistance to the antibiotic, ampicillin. "arac" Gene which codes for AraC, a protein which regulates production of the Green Fluorescent Protein. Gene Regulation One of the main instructional highlights of the pglo Transformation Kit was the concept of gene regulation. In this kit, the bacteria which were transformed with the pglo plasmid were plated onto LB/amp and LB/amp/ara agar plates. Expression of the GFP gene is under the regulatory control of the arabinose promoter. Thus, when the bacteria were plated onto LB agar containing arabinose (LB/amp/ara), GFP was expressed and the colonies appeared bright green. Conversely, when the bacteria were plated onto LB agar that did not contain arabinose (LB/amp), the gene was turned off and the colonies appeared white. (See Appendix D for more details.) The concept of gene regulation can be further expanded upon in this kit. If a white colony (which contains the GFP gene, but is not expressing GFP in the absence of arabinose) is seeded into LB/amp/ara liquid media, the arabinose present in the media will be taken up by the bacteria, which subsequently turns on the GFP gene. Thus the students will be seeding a white 10

14 colony which has the dormant GFP gene into arabinose containing media. Arabinose turns on the dormant gene, resulting in a fluorescent green liquid culture. In addition, the student will be seeding a green fluorescent colony into LB/amp/ara liquid culture. In this case the gene will remain "on" and the liquid culture will also fluoresce bright green. Lesson 2 Inoculation Growing a Cell Culture Isolation of Single Bacterial Colonies In this activity, students will pick one white colony from their LB/amp plates and one green colony from their LB/amp/ara plates for propagation in parallel liquid cultures. Cloning describes the isolation and propagation of a single gene. Because a single bacterial colony originates from a single bacterium, all the individual bacteria in the colony are genetically identical and they are called clones. When the students pick colonies (or clones) of both green and white bacteria from their agar plates, single isolated colonies are chosen for transfer to the culture media. Single isolated colonies which are separated from other colonies on the agar by at least 1 2 millimeters are generally not contaminated with other bacteria. The schematic below illustrates the genes expressed in fluorescent green colonies. Green fluorescent colonies ( ) Transformed bacterial cell Green fluorescent protein (GFP) The instructor may want to demonstrate how to scoop a single, well-isolated colony from the agar using an inoculation loop. Again, it is very important that proper sterile technique is followed during the picking of colonies and subsequent dissociation of the colonies into the culture tubes. Remind students to keep their plates covered and their tubes capped wherever possible. Overnight Liquid Cultures The GFP gene requires a 32 C (or lower) incubation temperature for optimal protein folding and fluorescence. If a 32 C incubator is unavailable, the bacteria can be cultured by shaking at room temperature, but this may require a 48 hour, rather than a 24 hour, culture period. In this lesson, liquid bacterial cultures will yield sufficient growth and production of protein simply by incubating overnight at 32 C. However, vigorous shaking of the liquid culture delivers more oxygen to the dividing cells allowing them to grow faster and produce more GFP. For this reason, we strongly recommend that a shaking incubator is used. If a shaking incubator is not available, cell cultures can be shaken manually for 30 seconds then incubated 1 2 days at 32 C. Simply shake the capped culture tubes vigorously for 30 seconds like a can of 11 Antibiotic resistance protein Bacterial proteins GFP regulator protein

15 spray paint. Then place the tubes on their sides in the incubator oven for 1 2 days. Placing the tubes horizontally in the oven maximizes the culture surface area and oxygen exchange. Removing the tubes and shaking them periodically during the incubation period will enhance cell growth further. The more constant and more vigorous the shaking, the more growth and the more production of the Green Fluorescent Protein. In either case, following incubation, the liquid culture should fluoresce bright green upon exposure to UV light. Culture Condition Days Required 32 C shaking 1 day 32 C no shaking 1 2 days* Room Temperature shaking 2 days Room Temperature no shaking Not recommended Lesson 3 Purification Phase 1 Bacterial Concentration and Lysis Centrifugation Centrifugation is a technique used to separate molecules on the basis of size by high speed spinning (somewhat analogous to the spin cycle in a washing machine where the clothes become compacted against the walls of the washer). In this lab session, the heavier bacterial cells will be separated from the liquid growth media by a single centrifugation step. Centrifugation results in a "pellet" of bacteria found at the bottom of the tube, and a liquid "supernatant" that resides above the pellet. The collection and concentration of bacteria is a first step in the isolation of GFP from the bacteria that were grown in the liquid media in Lesson 2. Using a disposable pipette, students transfer the 2 milliliters of culture from the culture tube into a 2 milliliters microtube in one gentle squirt. They will then spin the bacterial pellet down to the bottom of the microtube and pour off (discard) the liquid supernatant above the pellet. The pellet will fluoresce bright green upon exposure to UV light because the green protein is being expressed within the bacteria. At this point, this short discussion of a "pellet" and "supernatant" may be helpful. Supernatant ori arac bla GFP Transformed bacterial cell Green fluorescent protein (GFP) Bacterial pellet Antibiotic resistance protein Bacterial proteins GFP regulator protein 12

16 Resuspension of the Bacterial Pellet In this step of the protocol, it is essential that the entire bacterial pellet is resuspended in TE Buffer. The students should add 250 µl of TE Buffer to the pellet of bacteria, and then resuspend the pellets by carefully, but rapidly, pipetting the cells up and down with the 250 µl volume of buffer. (If vortexes are available, gentle vortexing will also aid in resuspension.) The volume in the tube can be visually inspected for large chunks of unresuspended bacteria. If chunks are observed, the students should continue to pipette up and down to fully resuspend the bacteria. Lysozyme Lysozyme is an enzyme that functions to degrade (or lyse) the bacterial cell wall, by cleaving polysaccharide (sugar) residues in the cell walls. The subsequent freeze-thaw step used in this lesson aids in the complete disruption of the wall and internal membrane. Complete disruption or "lysis" releases soluble components, including GFP. Lysozyme is naturally found in human tears, acting as a bactericidal agent to help prevent bacterial eye infections. Lysozyme gets its name from its natural ability to "lyse" bacteria. Lesson 4 Purification Phase 2 Removing Bacterial Debris This final centrifugation step serves to separate the large particles of lysed bacteria (such as the cell membrane and walls) from the much smaller proteins, including GFP. The larger bacterial debris are pelleted in the bottom of the microtube, while the smaller proteins remain in the supernatant. At this stage, the supernatant will fluoresce bright green upon exposure to UV light. Students should then, very carefully, remove the supernatant from the pellet and place it into a new 2.0 milliliter microtube using a clean 1 milliliter pipette. This should be done immediately to prevent pellet debris from leaching and contaminating the supernatant and potentially clogging the chromatography column in the purification procedure of lesson 5. Freezing + + Lysozyme centrifugation Resuspended bacterial pellet Lesson 5 Purification Phase 3 Protein Chromatography Chromatography is a powerful technique for separating proteins in a complex mixture. Bacteria contain thousands of endogenous proteins from which GFP must be separated. In chromatography, a cylinder, or column is densely filled with a "bed" of microscopic beads. These beads form a matrix through which proteins must pass before being collected. The matrix the students will employ in this case has an "affinity" for the molecule of interest (GFP), but not for the other bacterial proteins in the mixture. GFP "sticks" to the column, allowing it to be separated from the bacterial contaminants. 13 Bacterial pellet (cell wall and debris)

17 Hydrophobic ("water hating") substances do not mix well with water. When they are dropped into salty water they tend to stick together. Some of the amino acids that make up proteins are very hydrophobic. In salt water, these parts of a protein tend to stick tightly to other hydrophobic substances. High salt causes the three dimensional structure of the protein to actually change so that the hydrophobic regions of the protein are more exposed on the surface of the protein and the hydrophilic ("water-loving") regions are more shielded. A chromatography column which is packed with hydrophobic beads is called a hydrophobic interaction matrix. When the sample is loaded onto the matrix in salt water, the hydrophobic proteins in the sample will stick to the beads in the column. The more hydrophobic they are the more tightly they will stick. When the salt is removed, the three dimensional structure of the protein changes again so that the hydrophobic regions of the protein now move to the interior of the protein and the hydrophilic ("water-loving") regions move to the exterior. The result is that the hydrophobic proteins no longer stick to the beads and drip out the bottom of the column, separated from the other proteins. Hydrophobic Interaction Chromatography (HIC) In lessons 4 and 5, the soluble GFP in the supernatant is purified using hydrophobic interaction chromatography (HIC). GFP has several stretches of hydrophobic amino acids, which results in the total protein being very hydrophobic. When the supernatant, rich in GFP, is passed over a HIC column in a highly salty buffer (Binding Buffer), the hydrophobic regions of the GFP stick to the HIC beads. Other proteins which are less hydrophobic (or more hydrophilic) pass right through the column. This single procedure allows the purification of GFP from a complex mixture of bacterial proteins. Loading the GFP supernatant onto the chromatography column When students load the GFP supernatant onto their columns, it is very important that they do not disturb the upper surface of the column bed when performing the chromatography procedure. The column matrix should have a relatively flat upper surface. A slightly uneven column bed will not drastically affect the procedure. However, subsequent steps of loading, washing, and eluting should minimize disrupting the column such that beads "fluff up" into the buffer. When loading the GFP supernatant onto the column, the pipette tip should be inserted into the column and should rest against the side of the column. The supernatant should be slowly expelled from the pipette, down the walls of the column. When the supernatant has completely entered the column, a green ring of fluorescence should be visible at the top of the bed when viewed with the UV light. There are four different buffers which are used in the HIC procedure. Each buffer should be slowly pipetted down the side of the column to minimize disturbance to the column resins. Equilibration buffer A medium salt buffer (2 M (NH 4 ) 2 SO 4 ) which is used to "equilibrate" or "prime" the chromatography column for the binding of GFP. Binding buffer An equal volume of high salt Binding Buffer (4 M (NH 4 ) 2 SO 4 ) is added to the bacterial lysate. The end result is that the supernatant containing GFP has the same salt concentration as the equilibrated column. When in a high salt solution, the hydrophobic regions of proteins are more exposed and are able to interact with and bind the hydrophobic regions of the column. Wash buffer A medium salt Wash Buffer (1.3 M (NH 4 ) 2 SO 4 ) is used to wash weakly associated proteins from the column; proteins which are strongly hydrophobic (GFP) remain bound to the column. When the Wash Buffer is applied to the column, care should again be taken to minimize disturbance of the column resin. Slow, steady pipetting down the side of the column is most 14

18 effective. At this point "a ring" of GFP should begin to penetrate the upper surface of the matrix (~ 1 2 mm into the bed). Elution buffer A low salt buffer (TE Solution; 10 mm Tris/EDTA) is used to wash GFP from the column. In low salt buffers (which have a higher concentration of water molecules), the conformation of GFP changes so that the hydrophilic residues of GFP are more exposed to the surface, causing the GFP to have a higher affinity for the buffer than for the column, thereby allowing the GFP to wash off the column. The 0.75 milliliters of TE (Elution) Buffer is applied to the column gently, as described above. The TE Solution will disrupt the hydrophobic interactions between the GFP and the column bed, causing GFP to let go and "elute" from the column. The GFP should pass down the column as a bright green fluorescent ring. This is easily observed using the UV light. If the column bed was disturbed in any of the preceding steps, the GFP will not elute as a distinct ring, but will elute with a more irregular, distorted shape. However, elution should still occur at this step. If successful, collection tube 3 should fluoresce bright green. Storage of tubes All of the collection tubes and their constituents can be parafilmed or covered and stored for approximately 1 2 weeks in the refrigerator. Important hints for successful chromatography 1. Place the column gently into the collection tubes. Jamming the column tightly into the collection tubes will create an air tight seal and the sample will not flow through. You can create a paper crutch by folding a small piece of paper, about the size of a match stick, and wedging it between the column and the collection tube. This crutch makes it impossible for an air tight seal to form, and insures that the column will flow. 2. The flow rate of the column can be increased in the elution step by placing the top cap tightly back onto the column. This creates air pressure which pushes on the column bed, causing the sample to flow faster. 3. The columns are designed to drip slowly. The entire chromatography procedure should take 20 to 30 minutes. It is important not to remove the column more than needed from collection tube to collection tube, as motion can cause major disturbance to the column bed. Binding Wash Elution 15

19 GFP Purification Quick Guide Lesson 2 Inoculation Growing Cell Cultures 1. Remove the transformation plates from the incubator and examine using the UV light. Identify several green colonies that are not touching other colonies on the LB/amp/ara plate. Identify several white colonies on the LB/amp plate. LB/amp/ara LB/amp 2. Obtain two culture tubes containing the growth media LB/amp/ara. Label one "+" and one "-". Using a sterile loop, lightly touch the loop to a green colony and immerse it in the "+" tube. Using a new sterile loop, repeat for a white colony and immerse it in the "-" tube (it is very important to pick only a single colony). Spin the loop between your index finger and thumb to disperse the entire colony. LB/amp/ara + LB/amp - 3. Cap the tubes and place them in the shaking incubator or on the shaking platform and culture overnight at 32 C or 2 days at room temperature. or Cap the tubes and shake vigorously by hand. Place in the incubator horizontally at 32 C for hours. Remove and shake by hand periodically when possible. + - Incubate at 32 C overnight or 2 days at room temperature 16

20 Lesson 3 Purification Phase 1 Bacterial Concentration 1. Label one microtube "+" with your name and class period. Remove your liquid cultures from the shaker and observe with the UV light. Note any color differences between the two cultures. Using a new pipette, transfer 2 ml of "+" liquid culture into the + microtube. Spin the microtube for 5 minutes in the centrifuge at maximum speed. The pipette used in this step can be repeatedly rinsed in a beaker of water and used for all following steps of this laboratory period. 2 ml Pour out the supernatant and observe the pellet under UV light Using a rinsed pipette, add 250 µl of TE solution to the tube. Resuspend the pellet thoroughly by rapidly pipetting up and down several times. 250 µl TE 4. Using a rinsed pipette, add 1 drop of lysozyme to the resuspended bacterial pellet to initiate enzymatic digestion of the bacterial cell wall. Mix the contents gently by flicking the tube. Observe the tube under the UV light. 1 drop lysozyme Freezer 5. Place the microtube in the freezer until the next laboratory period. The freezing causes the bacteria to rupture completely. 17

21 Lesson 4 Purification Phase 2 Bacterial Lysis 1. Remove the microtube from the freezer and thaw using hand warmth. Place the tube in the centrifuge and pellet the insoluble bacterial debris by spinning for 10 minutes at maximum speed. Thaw Centrifuge 2. While your tube is spinning, prepare the chromatography column. Remove the cap and snap off the bottom from the prefilled HIC column. Allow all of the liquid buffer to drain from the column (~3 5 minutes). 3. Prepare the column by adding 2 ml of Equilibration Buffer to the top of the column. This is done by adding two 1 ml aliquots with a rinsed pipette. Drain the buffer to the 1 ml mark on the column. Cap the top and bottom and store the column at room temperature until the next laboratory period. Equilibration buffer (2 ml) 1 ml 4. After the 10 minute spin, immediately remove your tube from the centrifuge. Examine the tube with the UV light. Using a new pipette, transfer 250 µl of the "+" supernatant into a new microtube labeled "+". Again, rinse the pipette well for the rest of the steps of this lab period µl 250 µl 5. Using a well rinsed pipette, transfer 250 µl of binding buffer to the "+" supernatant. Place the tube in the refrigerator until the next laboratory period. 18

22 Lesson 5 Purification Phase 3 Protein Chromatography 1. Label 3 collection tubes 1 3 and place the tubes in the foam rack or in a rack supplied in your laboratory. Remove the caps from the top and bottom of the column and place the column in collection tube 1. When the last of the buffer has reached the surface of the HIC matrix proceed to the next step below. 2. Using a new pipette, carefully and gently load 250 µl of the + supernatant onto the top of the column. Hold the pipette tip against the side of the column wall, just above the upper surface of the matrix and let the supernatant drip down the side of the column wall. Examine the column using a UV light. Note your observations. After it stops dripping transfer the column to collection tube 2. 1 (250 µl) + supernatant + 1 Wash buffer (250 µl) 3. Using the rinsed pipette, add 250 µl of wash buffer and let the entire volume flow into the column. Examine the column using the UV light. Note your observations. After the column stops dripping, transfer it to tube 3. 2 TE buffer (750 µl) 4. Using the rinsed pipette, add 750 µl of TE Buffer and let the entire volume flow into the column. Examine the column using the UV light. Note your observations Examine all three collection tubes and note any differences in color between the tubes. Parafilm or Saran Wrap the tubes and place in the refrigerator until the next laboratory period

23 Teachers Answer Guide Lesson 1 Review Questions 1. Proteins a. What is a protein? A macromolecule which consists of chains of amino acids. b. List three examples of proteins found in your body. Antibodies, digestive enzymes, hair proteins, hormones, and hemoglobin are all examples of proteins. c. Explain the relationship between genes and proteins. Genes contain the genetic code which determine the amino acid composition of a protein. There is a unique gene for each protein within all of the cells of the body. 2. Using your own words, define or describe cloning. The duplication and propagation of a cell or organism. 3. Describe how bacterial cells in a "library" are different from the cells of a single colony. A library of bacterial cells contains a diverse mixture of bacterial types which contains a diverse mixture of genes. A single colony of bacteria originates from an individual clone which only contains a single gene. 4. Describe how you might recover the cancer-curing protein from the bacterial cells. One can isolate a single fluorescent green colony of bacteria and grow large amounts of the bacteria in a liquid growth media. Bacteria in liquid media can be concentrated by centrifugation. After the bacterial cells are lysed to release the cancer curing protein, the protein can be isolated by passage through a chromatography column which has an affinity for the cancer-curing protein. 20

24 Lesson 2 Review Questions 1. What is a bacterial colony? A bacterial colony is a large group or cluster of bacterial cells that originated from a single, clonal cell. 2. Why did you select one green and one white bacterial colony from your agar plate(s)? What could this prove? A green colony was picked in order to propagate or produce large amounts of the green cancer-curing protein. A white colony was chosen as a "negative control" to show that only the green colonies contain and produce the green cancer-curing protein. 3. How are these items helpful in this cloning experiment? a. ultraviolet (UV) light The green protein fluoresces, and is thus visible, when exposed to the UV light. b. incubator The incubator provides a warm temperature which enables the bacteria to grow. c. shaking incubator A shaking incubator provides a warm temperature and provides aeration which oxygenates the bacterial cultures. Increased oxygen accelerates the growth rate of bacteria. 4. Can you predict what would happen if you took one of the green colonies from the LB/amp/ara plate and streaked it onto an LB/amp plate? Conversely, what would happen if you took a white colony from the LB/amp plate and streaked it onto an LB/amp/ara plate? Explain your answer. If a green colony was streaked onto an LB/amp plate, the resulting colonies would be white. This plate does not contain arabinose which is needed to induce expression of the GFP gene and generate green fluorescent colonies. If a white colony was streaked onto an LB/amp/ara plate, the resulting colonies would be green. This plate contains arabinose which induces expression of the GFP gene and generates green fluorescent colonies. 21

25 Lesson 3 Review Questions 1. You have used a bacterium to propagate a gene that produces a green fluorescent protein. Identify the function of these items you used in Lesson 3. a. Centrifuge Functions to pellet the bacteria and separate the bacteria from the growth media. b. Lysozyme Functions to enzymatically digest the bacterial cell well, which in turn weakens the cell wall so that it will rupture upon freezing. c. Freezer Functions to freeze the bacteria which causes the cytoplasm to expand, which completely ruptures the weakened cell wall. 2. Can you explain why both liquid cultures fluoresce green? The green colony seeded into the (+) tube fluoresces green because the arabinose in the liquid culture media continues to induce expression of the GFP gene, which results in a green culture. The white colony seeded into the (+) tube fluoresces green because the arabinose in the liquid culture media turns on expression of the GFP gene which was previously off on the LB/amp plate (which lacks arabinose), which results in a green culture. 3. Why did you discard the supernatant in this part of the protein purification procedure? The supernatant contains the bacterial growth media and does not contain the desired GFP. 4. Why did you discard the white liquid from the "-" tube but keep the green one? The white culture of bacteria does not contain the GFP and is not needed for the subsequent purification step. 5. Can you explain why the bacterial cells' outer cell wall ruptures when the cells are frozen? What happens to an unopened soft drink when it freezes? When a bacterial cell freezes, the volume of cytoplasm expands. The expansion puts pressure on the weakened cell wall, which then ruptures from the pressure. 6. What was the purpose of rupturing or lysing the bacteria? The bacteria need to be ruptured in order to release the GFP, which can then be purified using column chromatography. 22

26 Lesson 4 Review Questions 1. What color was the pellet in this step of the experiment? What color was the supernatant? What does this tell you? The pellet should be a whitish or pale green color. The supernatant should fluoresce bright green. The fluorescent green color of the supernatant indicates that the green protein was released from the bacteria and remained in the supernatant. The much lighter color of the bacterial pellet suggests that the GFP was released from the bacteria upon lysis. 2. Why did you discard the pellet in this part of the protein purification procedure? The pellet contains unwanted bacterial debris bacterial cell walls, membranes, and chromosomal DNA. The pellet contains little, if any, GFP and can be discarded. 3. Briefly describe hydrophobic interaction chromatography and identify its purpose in this lab. Protein chromatography is a technique which can be used to separate or purify proteins from other molecules. This lab used hydrophobic interaction chromatography to purify GFP based upon its hydrophobic properties. 23

27 Lesson 5 Review Questions 1. List your predictions and observations for the sample and what happens to the sample when the following buffers are added to the HIC column. Observations Under UV Light Collection Tube Number Prediction (column and collection tube) Tube 1 GFP should stick to the GFP resides as a band at Sample in Binding Buffer column the top of the column Tube 2 GFP should stick to the GFP remains as a broad Sample in Wash Buffer column band on top of the column Tube 3 GFP should elute from the GFP travels down the column Sample with Elution Buffer column as a ring and elutes into tube 3 2. Using the data table above, compare how your predictions matched up with your observations for each buffer. a. Binding Buffer GFP binds to the top of the chromatography column. b. Wash Buffer GFP remained bound to the top of the column. c. Elution Buffer GFP is eluted from the column. 3. Based on your results, explain the roles or functions of these buffers. Hint: how does the name of the buffer relate to its function? a. Equilibration Buffer This buffer prepares the column for the application of GFP. Equilibration buffer raises the salt concentration of the column to match that of the bacterial GFP lysate. b. Binding Buffer This buffer raises the salt concentration of GFP which causes a conformational change in GFP, exposing the hydrophobic regions. c. Wash Buffer Wash buffer functions to wash away less hydrophobic, contaminating proteins from the column. d. TE (elution) Buffer This buffer functions to remove GFP from the column. 4. Which buffers have the highest salt content and which have the least? How can you tell? Binding Buffer >>Equilibration buffer>>wash buffer>>elution buffer Binding buffer has the highest concentration of salt because it is needed to raise the salt concentration of the GFP lysate. The hydrophobic patches of proteins are exposed in high salt buffer. TE elution buffer has the lowest salt concentration because it causes GFP to elute from the column. The hydrophobic patches of proteins re-orient to the interior, and the hydrophilic regions are exposed in low salt buffer. 24

28 5. Were you successful in isolating and purifying GFP from the cloned bacterial cells? Identify the evidence you have to support your answer. If tube 3 fluoresces green, the student was successful in purifying GFP. If GFP is not present in tube 3, examine the column application of an incorrect buffer would prevent the elution. Alternatively, if the student did not start with a bright green culture, then tube 3 will not be extremely bright. 25

29 Green Fluorescent Protein (GFP) Purification Student Manual "Bioengineered DNA was, weight for weight, the most valuable material in the world. A single microscopic bacterium, too small to see with the human eye, but containing the gene for a heart attack enzyme, streptokinase, or for "ice-minus" which prevented frost damage to crops, might be worth 5 billion dollars to the right buyer." Michael Crichton - Jurassic Park Contents Lesson 1 Genetic Transformation Review Finding the Green Fluorescent Molecule Lesson 2 Inoculation Growing a Cell Culture Lesson 3 Purification Phase 1 Bacterial Concentration and Lysis Lesson 4 Purification Phase 2 Removing Bacterial Debris Lesson 5 Purification Phase 3 Protein Chromatography 26